REVERSAL OF DIABETES INDUCED SPLENIC MITOCHONDRIAPATHY BY
AQUEOUS EXTRACTS OF Musa parasidisiaca and Psidium guajava LEAF IN MALE
ALBINO RATS
BY
OLAOYO MICHAEL OLAWALE
20170998
A RESEARCH PROJECT
SUBMITTED TO THE
DEPARTMENT OF BIOCHEMISTRY,
COLLEGE OF BIOSCIENCES,
FEDERAL UNIVERSITY OF AGRICULTURE, ABEOKUTA.
IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF
BACHELOR OF SCIENCE (B. Sc.) DEGREE IN BIOCHEMISTRY.
DECEMBER ,2021.
CERTIFICATION
This research titled “Reversal of diabetes induced splenic mithochondriopathy by aqueous
extracts of Musa parasidiaca and Psidium guajava in male albino rats by OLAOYO, Michael
Olawale, with matriculation number 20170998 meets the regulation for the award of the degree of
Bachelor of Science (B. Sc.) degree in Biochemistry in the Federal University of Agriculture.
Abeokuta and is approved for its contribution to scientific knowledge and literary presentation.
OLAOYO, Michael Olawale
DATE
Student
PROF. O. ADEMUYIWA
DATE
Supervisor
DR. J. K. AKINTUNDE
Head of Department
DATE
DEDICATION
This project is dedicated to God Almighty ,the owner and the sustainer of my life, who is in his
infinite mercy has kept me till date and my parent Mr and Mrs Olaoyo who continually supported
me throughout this journey
ACKNOWLEDGEMENT
My utmost goes to the Almighty God for his love , sufficiency ,protection, mercy and grace to me
throughout my stay in the university and all ramifications of my life and for making everything
possible.
My sincere appreciation goes to my parents Mr and Mrs Olaoyo for their care, unquantifiable,
love, financial and spiritual support. I also want to appreciate my siblings who continually
supported me financially.
To my supervisor, Prof. Ademuyiwa thank you for your guidance, support, excellence and
supervision God bless you, sir. To my H.O.D ,Dr J.K Akintunde, and all my lecturers; Prof.(Mrs)R.
N. Ugbaja,O., Prof. Akinloye,O. A., Dr. Akamo,A. J., Dr. Eteng, E.O., Dr.Onunkwor, B.O., Dr.
Akintunde, J. K., Dr. Adeyi, Dr. Babayemi, D.O., Dr. Akinloye, D.I., Mr. Somade ,O.T., Mr.
Moses,C.A., Mr.Adeleye, Mr. Ogbonna,C. Thanks for impacting so much into to academically
and morally.
To my project mates Obasa Islamiat, Praise Adeiza, Jamiu Musbau, Akintola Joshua, Adeoti
Bolaji. I say a very big thank you for your cooperation and diligence towards making this project
a success.
ABSTRACT
The International Diabetes Federation reports that in 2015, Around 415 million people worldwide
have diabetes, and by 2040, that figure is projected to rise to over 640 million. Musa parasidisiaca
and Psidium guajava been reported to have antihyperglycemic effects. This study is aimed to
investigate the reversal of diabetes induced splenic mitochondriapathy by aqueous extracts of
Musa parasidisiaca and Psidium guajava leaf in male albino rat. Forty eight rats were divided into
eight groups (n = 6). Six animals were sacrificed to obtain the baseline data and served as group I.
Group II served as the control group and were given distilled water for 3days. Group III were
rendered diabetic by a single intraperitoneal injection of streptozotocin (STZ) (50mg/kg bwt.) and
sacrificed 3rd day diabetes was confirmed. Group IV were rendered diabetic intraperitoneally as
well as were given distilled water for 28days. Groups V and VI were rendered diabetic through a
single intraperitoneal injection of streptozotocin as well and were treated with 100mg/kg btw of
Musa parasidisiaca and Psidium guajava extracts respectively for 28days. Groups VII and VIII
were not diabetic but were given 100mg/kg btw and 300mg/kg btw of Musa parasidisiaca extract
respectively for 28days. The route of administration of these extracts is oral. The result showed
that oral administration of aqueous extract of Musa paradisiaca and Psidium guajava reversed the
diabetes induced splenic mitochondriopathy in male albino rats
Key words: Musa parasidisiaca, Psidium guajava, mitochondriapathy and streptozotocin
Keywords: Musa paradisiaca, Diabetes, Mitochondriopathy, Electrolyte disorder.
TABLE OF CONTENT
CERTIFICATION ........................................................................................................................................ 1
DEDICATION .............................................................................................................................................. 3
ACKNOWLEDGEMENT ................................................................ Ошибка! Закладка не определена.
ABSTRACT...................................................................................... Ошибка! Закладка не определена.
TABLE OF CONTENTS .................................................................. Ошибка! Закладка не определена.
LIST OF TABLES ............................................................................ Ошибка! Закладка не определена.
LIST OF FIGURES .......................................................................... Ошибка! Закладка не определена.
CHAPTER ONE ............................................................................... Ошибка! Закладка не определена.
INTRODUCTION ............................................................ Ошибка! Закладка не определена.
1.0
1.1 Diabetes...........................................................................Ошибка! Закладка не определена.
1.2
Spleen..............................................................................Ошибка! Закладка не определена.
1.3
Musa paradisiaca............................................................Ошибка! Закладка не определена.
1.4
Justification .....................................................................Ошибка! Закладка не определена.
1.5
Aim and Objectives of the Study ....................................Ошибка! Закладка не определена.
CHAPTER TWO .............................................................................. Ошибка! Закладка не определена.
2.0
2.1
LITERATURE REVIEW ................................................. Ошибка! Закладка не определена.
Diabetes...........................................................................Ошибка! Закладка не определена.
2.1.1 Signs and Symptoms .......................................................Ошибка! Закладка не определена.
2.1.2 Complications .................................................................Ошибка! Закладка не определена.
2.1.3 Diagnosis.........................................................................Ошибка! Закладка не определена.
2.1.4 Management ............................................................................................................................ 25
2.2
Spleen..............................................................................Ошибка! Закладка не определена.
2.2.1
Mechanism of diabetic spleen damage……………………….……………………..27
2.3 Mitochondria…………………………………………………………………..
2.4 ATPases…………………………………………………………………………..
2.4.1 Na+/K+ ATPase…………………………………………………………………………………………………………….
2.4.2 Ca2+/Mg2+ ATPase………………………………………………………………………………………………………
2.5 Musa parasidiaca……………………………………………………………………………………………………
2.5.1 Description of Musa paradisiaca………………………………………………………………………….
CHAPTER THREE .......................................................................... Ошибка! Закладка не определена.
3.0
MATERIALS AND METHODS ...................................... Ошибка! Закладка не определена.
3.1
Chemicals and Reagents .................................................Ошибка! Закладка не определена.
3.2
Apparatus and Equipment ....................................................................................................... 32
3.3
Preparation of Banana Stalk Extract ....................................................................................... 32
3.4
Experimental Animals.....................................................Ошибка! Закладка не определена.
3.5
Biochemical Assays ........................................................Ошибка! Закладка не определена.
3.6
Statistical Analysis ..........................................................Ошибка! Закладка не определена.
CHAPTER FOUR............................................................................. Ошибка! Закладка не определена.
4.0
4.1
RESULTS ......................................................................... Ошибка! Закладка не определена.
Blood Glucose Concentrations Of The Animals .............Ошибка! Закладка не определена.
4.2
Spleenic Ca2+/Mg2+ Atpase Activity Of The animals Ошибка!
Закладка
не
определена.
4.3
Spleenic Na+/K+ Atpase Activity Of The Animals .........Ошибка! Закладка не определена.
Spleenic Mg2+ Atpase Activity Of The
4.4
Animals…………………………………………Ошибка! Закладка не определена.
4.5Body Weight Gain/Loss Of The Animals ................................Ошибка! Закладка не определена.
4.6
Sometic Index Of The Spleen Of The Animals ..............Ошибка! Закладка не определена.
4.7 Water Intake Of The Animals………………………………………………………………………………………………..
4.8 Feed Intake Of The Animals………………..
CHAPTER FIVE .............................................................................. Ошибка! Закладка не определена.
5.0
DISCUSSION AND CONCLUSION ......................................................................................... 48
5.1
DISCUSSION ......................................................................................................................... 48
5.2
CONCLUSION ...............................................................Ошибка! Закладка не определена.
REFERENCES ................................................................................. Ошибка! Закладка не определена.
APPENDIX ....................................................................................... Ошибка! Закладка не определена.
LIST OF TABLES
Tables
Pages
1. Experimental Design…………………………………………………………39
2. Determination of Magnesium Concentration…………………………….......45
3. Determination of Calcium Concentration……………………………………47
4. Determination of Potassium Concentration………………………………….49
5. Effects of diabetes on weight gain/loss of the animals………….…………...54
LIST OF FIGURES
Figures
Pages
1. Morphological view of Musa paradisiaca tree…………………………........17
2. Structures of some phytochemicals present in Musa paradisiaca…………....20
3. A picture of the banana stalk used for the research…………………………..38
4. Plasma magnesium concentration of diabetes-induced rats…………………56
5. Plasma calcium concentration of diabetes-induced rats ……………….……57
6. Plasma sodium concentration of diabetes-induced rats…………………......59
7. Plasma potassium concentration of diabetes-induced rats ………………….60
8. Plasma Calcium-Magnesium ATPase activity of diabetes induced rats…….62
CHAPTER ONE
1.O.
INTRODUCTION
1.1.
Diabetes
Diabetes is a metabolic disorder characterized by hyperglycemia. It may be due to insufficiency in
secretion of endogenous insulin (type -1 diabetes) or insensitivity of the cells to be insulin action
(type -2 diabetes) (Maritim et al., 2002). This leads to the inability of the cell to convert excess
glucose in the body. This condition is termed as diabetes and it pave way for several complications
in the host cells and tissues. Although the etiology of this disease is not well defined, viral
infection, autoimmune disease,and environmental factors have been implicated in the study of the
disease (Maritim et al., 2002).
Increased oxidative stress is a widely accepted participant in the development and progression of
diabetes and its complications (Ceriello,2000). Diabetes is usually accompanied by increased
production of oxygen free radicals (OFRs)or impaired antioxidant defenses. Mechanisms by which
increased oxidative stress is involved in the diabetic complications are partly known, including
activation of transcription factors, formation of advanced glycated end product (AGEs), and
actication of protein kinase C (Maritim et al.,2002). OFRs have been implicated in both b-cells
destruction and as well as in liver injury(Roza et al., 1995).
Mammalian cells are equipped with both enzymic and non enzymic antioxidant defense to
minimize the cellular damage caused by interaction between cellular constituents and OFRs
(Freeman et al., 1982). OFRs are generated as by products of normal cellular metabolism;
however, several conditions are known to disturb the balance between OFR production and cellular
defense mechanisms. This imbalance can result in cell result in cell dysfunction and destruction
resulting in tissue injury. The increase in the level of OFRs in diabetes could be due to their
increased production and /or decreased destruction by non-enzymic and enzymic antioxidants.
Medicinal plants have been used as sources of medicine in virtually all cultures (Baquer 2005).
During the last decade, the use of traditional medicine has expanded globally and is gaining
popularity. It has continued to be used not only for primary health care medicine is predominant
in the health care system (Lanfranco 1999).
More than 400 plant species have hypoglycemic activity have been available in literature. Many
of these medicinal plants contain large amounts of antioxidants such as polyphenols , which can
play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet
oxygen, or decomposing peroxide (Neelesh et al.,2010). Many of these phytochemicals possess
significant antioxidant capacities that are associated with lower occurrence and lower mortality
rates of several human diseases.
1.2 SPLEEN
The spleen is an organ located in the upper left side of the abdomen that plays an important role
in the body's immune system and the production of blood cells. The spleen acts as a filter for the
blood, removing old or damaged red blood cells and producing white blood cells, which help fight
off infections (Ganong, 2005). The spleen also helps regulate the amount of blood in the body, and
it stores and releases red blood cells and platelets as needed (National Institutes of Health, 2017).
1.3.
Musa paradisiaca
The essence of medicinal plants cannot be over emphasized even in this present era. Medicinal
plants are chief sources of chemical substances (i.e phytochemicals with prospective therapeutic
effects (Mukerjee et al., 1998). According to the World Health Organization (WHO), they are
mostly used in traditional medicine as herbal remedies for varieties of disease throughout the
world , exceeding conventional drugs (Evans 1994; Palombo 2005). Majority of the worlds
population residing in rural areas of under developing and developing countries rely mostly on
medicinal plants for primary healthcare due to its affordability and accessibility particularly in the
absence of modern medical facilities (Vijai et al., 2015).
Musa paradisiaca (plantain) is a staple perennial tree cultivated in the tropics for its carbohydrate
content and be consumed both ripe and unripe (Ahenkora et al., 1997). It is an herbaceous plant
witt a robust , succulent and very juicy tree like pseudo stem which can attain a height 20 to 25 ft.
growing from a fleshy rhizome (corm), a crown of large elongated oval elliptical deep green leaves
with prominent mid ribs (Mohammad and Saleha, 2011; Lavanya et al ., 2016).
The various parts of Musa paradisiaca possess different therapeutic effects; the fruits are
used traditionally in treatment of diarrhea (unripe), dysentery, intestinal lesions in ulcerative
colitis, diabetes (unripe), in sprue uremia ,nephritis, gout, hypertension, cardiac disease. The
leaves (ashes) are used in eczema, the stem juice of fruited plant is used for treating diarrhea ,
dysentery, cholera, otalgia ,haemoptysis and the flower is used in dysentery, diabetes and
menorrhagia (Ghani,2003;Khare ,2007; Okoli, 2007).
1.4 Psidium guajava L.
Worldwide, medicinal plants and their bioactive components are utilized to treat a wide range of
illnesses. According to reports, over 80% of people use medicinal plants or their bioactive
substances to prevent, treat, or manage a variety of disorders (Joshi et al., 2013). Many scientists
and researchers have recently become interested in the uses of medicinal plants or their biologically
active compounds due to their use in the development of new drugs or the identification of natural
therapeutic component (Dimmito et al., 2021) and in ethnomedicinal uses for the treatment of lifethreatening diseases such as cancer, diabetes, and hypertension (Sofowora et al., 2013, WHO.,
2019).
One of the herbs used in traditional medicine for the treatment of many ailments is Psidium
guajava. Guava is the common name for Psidium guajava L. It is a tropical food plant and shrub
tree in the Myrtaceae family (Ravi and Divyashree, 2014). It has a maximum height of 10 m and
is found in many different nations. Psidium guajava is an economically significant food plant with
a variety of therapeutic benefits It has a thin trunk with peeling, smooth, and uneven bark. The
thick, dark-green leaves have noticeable veins ( Naseer et al., 2018). It blooms in white, and the
fruit is filled with pulp and tiny, firm seeds (Morais-Braga et al., 2016).
The various components of P. guajava, including the stem, bark (Beidokhti et al., 2020), fruits,
leaves, and roots (Weli et al., 2019), are used in ethnomedicine to treat conditions like diarrhea,
rheumatism, and diabetes, as well as digestive issues, laryngitis, ulcers, malaria, cough, and
bacterial infections (Ravi and Divyashree, 2014), wound healing and pain relief (Metwally et al.,
2010). Depending on the type of ailments, many natives use decoction, infusion, and/or boiled
preparations of P. guajava topically or orally (Daz-de-Cerio et al., 2017). For instance, P. guajava
leaves can be applied topically to wounds while aqueous leaf extract can be taken internally by
diabetics individual to lower their blood glucose levels (Gutierrez et al., 2008).
According to Gutierrez et al. (2008) and Weli et al. (2019), Psidium guajava contains essential
chemical components such flavonoids, tannins, phenols, alkaloids, triterpenes, saponins,
carotenoids, lectins, vitamins, carbohydrate, fiber, fatty acids, and glycosides. Guaijaverin,
quercetin, kaempferol, apigenin, catechin, chlorogenic acid, hyperin, gallic acid, epicatechin,
myricetin, caffeic acid, and epigallocatechin gallate are just a few of the many beneficial phenolic
compounds found in the leaves (Kumar et al., 2021).
1.5 MITOCHONDRIA
Mitochondria, which are double-membrane-bound organelles, are found in the majority of
eukaryotic organisms and are known as the cell's powerhouse due to their primary role in
producing adenosine triphosphate (ATP) as a source of chemical energy. In 1880, Albert von
Kolliker discovered them while studying the voluntary muscles of insects. Glucose transporters
GLUT-1 and GLUT-4 have different locations in various cell types, as reported by Hundal et al.
(1992), while Jackson et al. (1987) noted that the reduction in the incremental increase in plasma
glucose concentrations after oral glucose administration is similar to the reduction in fasting
plasma glucose concentrations. Besides controlling the cell cycle and promoting cell development,
mitochondria are also involved in signaling, cellular differentiation, and cell death, as reported by
Neuspiel M (2006). The number of mitochondria in a cell can vary significantly by organism,
tissue, and cell type, with liver cells having over 2000 mitochondria and mature red blood cells
having none. The mitochondrion is composed of compartments with specific functions, including
the outer membrane, intermembrane space, inner membrane, cristae, and matrix.
1.4. JUSTIFICATION OF STUDY
Since none of the antidiabetic drugs could give a long- term glycemic control without producing
any unfavorable side effects, medicinal plants which are effective in improving plasma glucose
level with minimal side effects are widely used in underdeveloped and developing countries as
an alternative therapy (Singh et al., 2007). Of these medicinal plants widely used in the field of
herbal medicine, Musa paradisiaca
(M. paradisiaca) has been reported to have many advantageous effects in the control of several
diseased conditions, including atherosclerosis, DM, hyperlipidemia, hypertension, and thyroid
dysfunctions (Parmar et al., 2007; Mallick et al., 2006) and also produces protective effects on
organs of the body, such as the kidneys, in certain clinical conditions (Vinaykumar et al., 2010).
(Vijayakumar et al., 2008) reported antioxidant efficacies of the isolated flavonoids from M.
paradisiaca in rats. The M. paradisiaca green fruit has been reported to have antihyperglycemic
effects due to stimulation of insulin production and glucose utilization (Ojewole et al., 2003) in
diabetic mice. Its elevated potassium (K) and sodium (Na) content has been correlated with the
glycemic state (Rai et al., 2009) Fibers from the M. paradisiaca fruit enhanced glycogenesis in the
liver and decreased fasting blood glucose concentration (Usha et al .,1989)
1.5. AIM AND OBJECTIVE OF THE STUDY
1.5.1 Aim of the study:
To investigate the effect of diabetes in liver mitochondria and possible reversal of these effects
using aqueous extract of musa paradisiacal stalk and guava leave extract in male albino rats
1.6. SPECIFIC OBJECTIVE




To investigate the antihyperglycemic effect of Musa paradisiacal and Psidium guajava on
the splenic mitocghondria of the male albino rat
To determine the effect of diabetes , musa paradiciaca stalk extract and guava leave extract
on liver mitochondrial activities of (Ca2+/Mg2+, Na+-K+-Mg2+ and Mg2+ ) ATPases.
To evaluate the body weight change effected by diabetic condition and possible reversal
by the treatment.
To determine the somatic index of the rats.
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 DIABETES
Polyuria (excessive urination), polydipsia (excessive thirst), and polyphagia are clinical outcomes
of hyperglycemia, a chronic, progressive metabolic condition associated with diabetes mellitus
(DM) (weight loss). Diabetes is a disease where either insufficient or unresponsive insulin
production occurs in the body. Diabetes is a problem with how glucose is metabolized, and its
impurity causes problems (Hannele Y.K, 1998). Maintaining normal glucose homeostasis is the
job of a glucose sensor found in the pancreatic beta-cell, which detects the increased release of
insulin. In contrast to a reduction in hepatic glucose output, higher amounts of circulating insulin
enhance the absorption of glucose by muscles and adipose tissue (Luciano and Meizhu, 1993).
While type 2 diabetes is of polygenetic origin and may have patients who may start with
hyperinsulinemia but have insulin resistance and through environmental factors such as diet and
sedentary lifestyle leads to an imbalance between glucagon and insulin levels, resulting in a
combination of causes toward hyperglycemia, type 1 diabetes typically has complete or nearly
complete knockout of insulin reserves, mediated solely by immunogenic responses from carriers
of certain genotypes. Due to hyperglycemia and specific elements of the insulin resistance
(metabolic) syndrome, people with Type 2 Diabetes Mellitus are at a high risk for both
microvascular complications (such as retinopathy, nephropathy, and neuropathy) and
macrovascular complications (such as cardiovascular comorbidities).
Non-communicable diabetes mellitus affects people in both established and developing nations.
The liver is just one of the many bodily systems that are impacted by this metabolic condition. The
metabolism of fats, carbohydrates, and proteins is affected by hyperglycemia, which is primarily
brought on by insulin resistance. Increased oxidative stress and an abnormal inflammatory
response, which trigger pro-apoptotic genes and harm hepatocytes, are the underlying mechanisms
by which diabetes causes liver damage. The accumulation of oxidative damage products in the
liver, such as malondialdehyde, fluorescent pigments, and conjugated dienes, is made worse by
the significant participation of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6, and
tumour necrosis factor.
Diabetes mellitus may aggravate liver disease by promoting fibrosis and inflammation through an
increase in mitochondrial oxidative stress, which is regulated by adipokines. Severe liver illness is
at risk in people with hereditary type 2 diabetes (CLD). El-serag et al. 2002, El-serag et al. 2004,
among others; Tolman et al.
2.1.1 SIGNS AND SYMPTOMS
The classic symptoms of untreated diabetes are unintended weight loss, polyuria (increased
urination), polydipsia (increased thirst), and polyphagia (increased hunger) (Cooke and Plotnick,
2008). Symptoms may develop rapidly (weeks or months) in type 1 diabetes, while they usually
develop much more slowly and may be subtle or absent in type 2 diabetes (WHO,2019). Several
other signs and symptoms can mark the onset of diabetes although they are not specific to the
disease. In addition to the known symptoms listed above, they include blurred vision, headache,
fatigue, slow healing of cuts, and itchy skin. Prolonged high blood glucose can cause glucose
absorption in the lens of the eye, which leads to changes in its shape, resulting in vision changes.
Long-term vision loss can also be caused by diabetic retinopathy. A number of skins rashes that
can occur in diabetes are collectively known as diabetic dermadromes (Rockefeller, 2015).
2.1.2 COMPLICATIONS
Long-term problems are more likely in all types of diabetes. These usually appear after many years
(10–20), but they might be the first symptom in people who haven't previously been diagnosed.
The main long-term issues are related to blood vessel injury. According to Sarwar et al. (2010),
diabetes doubles the chance of cardiovascular disease, and according to O'Gara et al. (2013),
coronary artery disease accounts for about 75% of deaths in diabetics. Stroke and peripheral artery
disease are two additional macro-vascular illnesses.
Damage to the eyes, kidneys, and nerves are the three main complications of diabetes brought on
by harm to small blood vessels. Damage to the blood vessels in the retina of the eye causes diabetic
retinopathy, which can cause progressive vision loss and other eye problems. Potential side effects
of diabetes include retinopathy, kidney, and neuropathy, which could lead to blindness (WHO,
2014). Additionally, diabetes increases the chance of developing cataracts, glaucoma, and other
eye conditions. People with diabetes are advised to see an eye specialist once a year. Diabetic
nephropathy, or harm to the kidneys, can cause tissue scarring, protein loss in the urine, and
ultimately chronic kidney disease, necessitating dialysis or kidney transplantation occasionally.
The most frequent side effect of diabetes is diabetic neuropathy, which causes damage to the body's
nerves (O'Gara et al., 2013). Numbness, tingling, pain, and altered pain perception are some of the
signs that can cause skin damage. Diabetes-related foot issues, such as diabetic foot ulcers, can
develop, are sometimes challenging to treat, and necessitate amputation on occasion. Additionally,
painful muscular atrophy and weakness are brought on by proximal diabetic neuropathy. Diabetes
and brain impairment are related. People with diabetes experience a 1.2 to 1.5-fold faster rate of
cognitive decline than people without the illness (Cukierman, 2005). Having diabetes, particularly
when taking insulin, increases an elderly person's risk of falling (Yang et al., 2016).
2.1.3 DIAGNOSIS
Using a blood test to measure blood glucose levels, diabetes mellitus is identified by showing any
one of the following symptoms:

Blood glucose measurement at fasting time 7.0 mmol/L (126 mg/dL). Blood is drawn for
this test after the patient has had enough time to fast overnight, i.e., in the morning before
meals.

Two hours after an oral glucose dose of 75 grams, as in a glucose tolerance test, plasma
glucose levels of less than 11.1 mmol/L (200 mg/dL) (OGTT)

Whether fasting or not, signs of elevated blood sugar and plasma glucose that are greater
than 11.1 mmol/L (200 mg/dL)

48 mmol/mol ( 6.5 DCCT%) of glycated hemoglobin (HbA1C).
2.1.4 MANAGEMENT
Diabetes management focuses on maintaining blood sugar levels as near to normal as possible
without depressing them. Usually, this can be done by making dietary adjustments, exercising,
losing weight, and taking the right medications (insulin, oral medications). It's crucial to educate
yourself on the condition and actively engage in treatment because complications are significantly
less prevalent and less severe in people with properly controlled blood sugar levels (Nathan et al.,
2005). The American College of Physicians states that a HbA1C reading of 7-8% is the target for
treatment (Qaseem et al., 2018). Other health issues that could hasten the negative impacts of
diabetes are also given consideration. These include obesity, metabolic syndrome, high blood
pressure, smoking, and lack of frequent exercise. Numerous people use specialized boots to
decrease the risk of ulcers in diabetic feet at risk, though the effectiveness of this strategy is still
unclear (Cavanagh, 2004).
DIABETES AND IONOREGULATORY DISTURBANCES
Diabetes mellitus is a complex metabolic and multifactorial disorder which results from the defect
in the shortage of insulin secretion or reduced insulin sensitivity (Wadkar et al., 2008). Type 1
diabetes results when the pancreatic b-cells fail to produce enough insulin, while type 2 diabetes
results from a condition known as insulin resistance, where sensitivity of the cells to the insulin
decreases. In rodents, type 1 diabetes can be induced by a single dose of streptozotocin (STZ)
injection (Junod et al.,2009), while type 2 diabetes can be induced either by three methods:
administration of nicotinamide followed by STZ induction (Szkudelski, 2012), feedingof high-fat
diet followed by a low-dose STZ injection (Skovso,2014) and STZ injection during neonatal
period (Portha et al.,2008). STZ is renowned for its selective pancreatic islet b-cell cytotoxicity
and has been extensively used to induce diabetes in animals (Papaccio et al., 2000). The induction
of STZ causes selective destruction of the b-cells of the islets of Langerhans through oxidative
stress-induced pathway (Szkudelski, 2001).
Electrolyte disturbances are common in patients with diabetes andmay be the result of an altered
distribution of electrolytes related to hyperglycemia-induced osmotic fluid shifts or of total-body
deficits brought about by osmotic diuresis. Complications from end-organ injury and the therapies
used in the management of diabetes may also contribute to electrolyte disturbances.
2.2 SPLEEN
The spleen is an organ located in the upper left side of the abdomen that plays an important role in
the body's immune system and the production of blood cells. The spleen acts as a filter for the
blood, removing old or damaged red blood cells and producing white blood cells, which help fight
off infections (Ganong, 2005). The spleen also helps regulate the amount of blood in the body, and
it stores and releases red blood cells and platelets as needed (National Institutes of Health, 2017).
The spleen is a vital organ in the human body that plays a crucial role in immune function, blood
filtration, and hematopoiesis. Located in the left upper quadrant of the abdomen, the spleen is a
highly vascularized organ that is divided into two distinct regions: the red pulp and the white pulp.
The red pulp of the spleen is responsible for filtering blood and removing damaged or old red
blood cells. The red pulp is composed of a network of blood vessels and sinuses that allow for the
efficient removal of cellular debris and pathogens from the bloodstream. Macrophages and
dendritic cells within the red pulp also play a critical role in the phagocytosis and removal of
foreign substances from the blood (Moseman, EA et all., 2007).
The white pulp of the spleen is responsible for the production of lymphocytes and the initiation of
immune responses. The white pulp is composed of lymphoid tissue and is subdivided into two
zones: the periarteriolar lymphoid sheath (PALS) and the follicles. The PALS is located around
central arteries within the spleen and is primarily composed of T lymphocytes, while the follicles
contain B lymphocytes and are organized around germinal centers (Cest, 2006).
In addition to its role in immune function, the spleen also plays a critical role in hematopoiesis, or
the production of blood cells. Hematopoietic stem cells (HSCs) are found within the red pulp of
the spleen and can differentiate into various types of blood cells, including red blood cells,
platelets, and white blood cells (Omatsu et all., 2010).
The anatomy of the spleen has been extensively studied, and several imaging modalities, including
ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), have been used
to visualize the organ. These techniques have allowed for the identification of various anatomical
features of the spleen, including its size, shape, and blood supply (Hatakeyama et all., 2012)
2.2.1 MEHANISM OF DIABETIC SPLEEN DAMAGE
One of the main ways in which diabetes damages the spleen is through oxidative stress. Oxidative
stress occurs when there is an imbalance between the production of reactive oxygen species (ROS)
and the antioxidant defense system. Several studies have shown that diabetes leads to an increase
in ROS production and a decrease in antioxidant enzyme activities in the spleen (Gao X et all.,
2008, Zhang L et all, 2014). This leads to damage to the spleen tissue, which can result in a
decreased ability to fight infections.
Another mechanism by which diabetes damages the spleen is through inflammation. Inflammation
is a normal response to infection or injury, but chronic inflammation can lead to tissue damage and
dysfunction. Diabetes is associated with increased levels of pro-inflammatory cytokines, such as
interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in the spleen (Zhang X et all., 2016,
Atkin XL et all., 2019). These cytokines can lead to the activation of immune cells in the spleen,
such as macrophages and T cells, which can cause tissue damage and dysfunction.
Additionally, diabetes can damage the spleen through alterations in the microvascular structure
and function. The microvasculature of the spleen is important for delivering oxygen and nutrients
to the cells and removing waste products. Several studies have shown that diabetes leads to changes
in the microvascular structure of the spleen, including thickening of the basement membrane and
a decrease in the number of capillaries. These changes can lead to decreased blood flow and tissue
hypoxia, which can result in tissue damage.
Diagram of the anatomy of the spleen (source: link.thought.co)
2.3 MITOCHONDRIA
The mitochondria, which are membrane-bound cell organelles, generate the majority of the
molecular energy needed to power a cell's metabolic processes (mitochondrion, singular). The tiny
molecule adenosine triphosphate acts as a reservoir for the chemical energy produced by the
mitochondria (ATP). Each small chromosome in a mitochondrial cell is unique. Mitochondria can
range in size from 0.5 to 10 m and typically have an oval or circular form. Mitochondria produce
heat, regulate cell growth and death, and store calcium for cell signaling tasks in addition to
supplying energy. In contrast to other cellular organelles, mitochondria have a distinct DNA, two
distinct membranes, and use binary fission for reproduction, suggesting that they may have
originated from prokaryotes (single-celled organisms).
Multipurpose organelles called mitochondria are inherited from the mother and form a massive
network in numerous cells. A complicated process comprising fission and fusion, mitochondrial
biosynthesis, and mitophagy maintains the balance of this network (Chan DC. 2012). Even though
they play a wide range of anabolic functions, mitochondria are best known for collecting and
preserving energy produced by the oxidation of organic substrates under aerobic conditions
through respiration. Oxygen serves as the final electron acceptor for the transfer of electrons from
NADH and FADH2 through the respiratory complexes I, II, III, and IV of MET. The energy
released during this process, which is held as a proton gradient, creates an electric potential across
the IMM. The F0F1 ATP synthase is activated by this membrane potential and generates ATP via
OXPHOS (respiratory complex V). The mitochondrial membrane potential, which also regulates
the influx of Ca2+ ions into the mitochondria to buffer cytoplasmic calcium, facilitates the import
of nuclear-encoded, mitochondrially targeted proteins (n-mitoproteins) (Wallace et al., 2010 ).
MET maintains low NADH/NAD+ ratios to support continuous glycolysis. A significant effect of
MET is reactive oxygen species (ROS), which operate in cell signaling pathways at low levels.
Strong antioxidant defense mechanisms in the mitochondria combat these free radicals to stop
oxidative damage to protein and lipids in higher quantities as well as to mitochondrial DNA
(mtDNA) (Chen et al., 2016). Additionally, mitochondria are involved in controlling apoptosis by
activating the mitochondrial permeability transition pore whenever ROS, the AMP/ATP ratio, and
Ca2+ levels in the mitochondria rise (Berridge et al., 2009).
Mitochondria play a vital role in bioenergetic and biosynthetic processes and can rapidly adapt to
the metabolic demands of the cell. Individual mitochondria join together to create dynamic
networks to meet demand when it is higher, whereas extra mitochondria are eliminated by
mitophagy and fission when the demand is lower (Palikaras et al., 2016). This degree of
adaptability to cellular needs is made possible by the nucleus and mitochondria's efficient
communication.
Diagram of the Mitochondria
2.5
ATPases
Adenosine triphosphate (ATP) is a class of enzymes that hydrolyzes a high energy phosphate link
to produce adenosine diphosphate. They capture the energy generated by the dissolution of the
phosphate link and use it to drive other cellular processes. Most of them are membrane-bound. The
membrane potential of a call at a specific moment is affected by the ATPases. By ensuring that
intracellular ions are under control, the ATPases and ion channels contribute to the maintenance
of the cell's health. Ion regulation and the preservation of the ion gradient across the cell membrane,
as was stated in Section 2.2 above, are crucial for maintaining cell homeostasis, and imbalances
may lead to pathological conditions. Misfunction of the ATPases has been linked to several
illnesses. They do this by acting as suitable biomarkers for organ performance (Abdulkareem et
al., 2019).
The V-type ATPase, P-type ATPase, and F-type ATPase are the three main kinds of ATPases. Ftype ATPases are active carriers that are involved in energy-saving processes in chloroplasts and
mitochondria. They aid in the protons' ascending transmembrane movement, which is fueled by
ATP hydrolysis. Since these ATPases were recognized as energy-coupling factors, the term "Ftype" has been used to describe them. Proton-transporting ATPases in the V-type family are
actually linked to F-type ATPases. They are in charge of acidifying intercellular spaces, hence the
letter V for vacuolar (Nelson and cox, 2005).
A big family of membrane proteins known as P-type ATPases is important for preserving cellular
homeostasis and producing the proper electrochemical gradients for cell survival. They mix the
active transport of cations or other substances across the membrane with the hydrolysis of ATP
(Maya-Hoyos et al., 2019). It was first described as a pump that was believed to be connected to
sodium ions in 1957 and was given its name based on the existence of an intermediate
phosphorylated process happening during catalyzing. The P-type ATPase structure consists of a
single subunit with the N- and C-termini exposed to the cytoplasm, several transmembrane
segments, four protein domains with highly conserved characteristics, and several transmembrane
segments. They have the same fundamental mechanism, as evidenced by this common structure
(Zhanget et al., 2020).
2.5.1 Na+/K+ ATPase
Jens Skou made the discovery of the Na+/K+ ATPase protein in 1957. The most notable membrane
protein that is expressed and integrated into the membrane is Na+/K+ ATPase. Almost all animal
tissues contain it. The components of the enzyme are and. The ASP369 residues in the enzyme
active site are phosphorylated when the larger -subunit (110 KDa) hydrolyzes ATP to ADP and
Pi. The enzyme then goes through an E1–E2 conformational shift, which results in
dephosphorylation of the enzyme. As a result of these processes, the energy released during ATP
hydrolysis promotes the electrogenic ion transfer of Na+ and K+ (3Na+ vs 2K+) across the
membrane. This indicates that the enzyme actively moves three Na+ ions outside the cell and three
K+ ions inside the cell against the gradient of their concentrations. Na+/K+ ATPase plays a crucial
role in the regulation of cellular physiology by maintaining an electrochemical gradient across
excitable tissues because it generates an electrophysiological gradient across the plasma and
mitochondria membrane (Kurauchi et al., 2019; Orlov et al., 2020).
2.5.2 Ca2+/Mg2+ ATPase
Using the energy from ATP hydrolysis, the membrane protein Ca2+/Mg2+ mediates the transfer
of Ca2+ and Mg2+ across the plasma or mitochondrial membrane against the electrochemical
gradient. Plasma membrane calcium ATPase (PMCA) and sarcoplasmic reticulum calcium
ATPase are two of the most significant Ca2+ regulatory systems (SERCA). The enzyme produces
a phosphorylated intermediate throughout the reaction cycles, making it a prime example of a Ptype ATPase. There are two primary conformations of the enzyme: E1 and E2. After intracellular
Ca2+ has bound to high affinity spots on E1, ATP can phosphorylate E1 and create the
intermediate E1P. The release of Ca2+ to the other side of the membrane is caused by the
conformational shift to E2P. A new pump cycle is enabled by the dephosphorylation of E2P to E2
and a new conformational change to E1 (de Sautu et al., 2018).
PSIDIUM GUAJAVA.L
Over 700 million people in sub-Saharan Africa depend on the tropical perennial plant Musa
paradisiaca (family: Musaceae), also known as plantain, whose extremely nutritious fruit is grown
there. It is typically grown for its caborhydrate content and can be eaten as a mature fruit or when
it is still an unripe fruit (ketiku A.O., 1973).. Bananas (Musa sapientum and Musa cavendishii) are
members of the Musaceae family and share the same growth pattern as plantains. However, they
vary from one another in terms of fruit color, stem and leaf color, and shape. Each stalk usually
produces one banana heart, also known as a single, male, sterile banana flower. The female flower
appears higher on the stem and gives birth to the actual flower without fertilization. fruit. The
banana is one of the fruits that is most commonly eaten worldwide. It is common knowledge that
bananas contain a number of vitamins, such as vitamin C, vitamin E, and beta-carotene.
(Sakakibara and Kanazawa, 2000).
The majority of people in the world, according to the World Health Organization, rely on
traditional medicine as a form of basic healthcare that uses plant extracts or their active
components (Lavanya et al.,2016). Indigenous information on the applications and uses of these
medicinal plants has been passed down from one generation to the next, aiding in the exploration
of different medicinal plants to uncover the scientific underpinnings of their traditional
applications. According to data provided by, the use of the biologically active components of
medicinal plants has been crucial in the discovery of novel chemical entities (Newman et al.,2003).
The leaves, roots, and flowers of the plant, among other components, have all been used
medicinally. In addition to being used as food, the fruit has also allegedly been used as an
antiscorbutic, apthrodisiac, and diuretic. Leaf juice is used to treat cuts, insect bites, and fresh
wounds. The plant's sap is used to treat diarrhea, dysentery, hysteria, and epilepsy (salawu et al.,
2010).
Using a banana stem extract from the Musaceae family as a treatment for kidney stone and high
blood pressure patients has been proposed. It has been discovered that taking a chloroform extract
of Musa sapientum flowers orally significantly reduces blood sugar and glycosylated hemoglobin,
increases total hemoglobin, and prevents weight loss (Pari and Uma-Maheswari, 1999). In
addition, the stem juice is used to treat hysteria, dysentery, and diarrhea, which are all nervous
illnesses. Fructose, xylose, galactose, glucose, and mannose are among the oligosaccharides that
bananas naturally contain, making them excellent prebiotics for the development of beneficial
bacteria in the colon (Gibson, 1998).
2.5.1
DESCRIPTION OF MUSA PARADISIACA
The herbaceous plant Musa paradisiaca, which can grow up to 9 meters tall, is indigenous to
Southeast Asia, India, Burma, the Malay Archipelago, New Guinea, Australia, Samoa, and tropical
Africa (Ahmed et al.,2006). It has a strong pseudostem that resembles a tree and a crown of
enormous, long, oval, deep green leaves with a prominent midrib that can measure up to 365 cm
in length and 61 cm in breadth. It has a small underground stem (corm) with buds, and from these
short rhizomes grow up to create a cluster of aerial shoots (suckers) that are near to the parent
plant. The adventitious roots are 75 cm long and extend 4-5 m laterally, , but primarily in the top
15 centimeters, where they create a dense mat. For stings and bites, the banana stalk sap can be
administered topically or consumed internally. The rhizome develops an inflorescence, or
blossom, when it is fully grown. It rises on an unbranched, smooth, tall stem that pierces the
pseudo-stem in the center, emerging at the summit in between the leaf cluster. Yellow flowers with
purple and red edging emerge in the summer on mature plants. The flower then changes into a
bunch of three to twenty hands, each with at least ten digits, that resembles a plantain.
Fig. 1: Morphological view of Musa paradisiaca tree
2.6.4 PHARMACOLOGICAL ACTIVITIES OF MUSA PARADISIACA.
Phytochemicals exert diverse pharmacological action when taken up into the body. The various
effects of Musa paradisiaca have been documented in both traditional and scientific literature and
has been subjected to research where more of its pharmacological effects has been unraveled. The
main pharmacological effects of this plant are:diuretics, shypoglycemic,antioxidant, analgesic and
anti diabetic effects (Vijai et al.,2015).

ANTI DIABETIC ACTIVITY
Santos et al.(2007) studied the anti diabetic effects of Musa paradisiaca by evaluating the effects
of Musa paradisiaca stem juice on blood glucose level(BGL)of normal and diabetic rats. The dose
of 500 mg/kg body weight produces a significant rise of 28.3% in blood glucose level after 6h of
oral administration in normal rats . Whereas in sub diabetic rats the same dose produces a rise of
16.4% in blood glucose level within 1h during glucose tolerance test (GTT)and a rise of 16% after
4h in fasting blood glucose levels of severe diabetic cases. This proves the anti diabetic activity of
Musa paradisiaca.

HYPOGLYCEMIC ACTIVITY
Musa paradisiaca juice exhibited hypoglycemic activity (Singhet al.,2007). Fibers from
M.paradisiaca fruit increased glycogensis in the liver and lowered fasting blood glucose,its green
fruits has been reported to exhibit hypoglycemic effects via stimulation of insulin production and
glucose utilization (Usha et al., 1989;Ojewole and Adewunmi 2003).

ANTIHYPERTENSIVE ACTIVITY
The aqueous extract of the ripe Musa Paradisiaca fruit was found to give a concentration dependent
hypotensive effect in noradernaline and potassium chloride contracted aortic rings isolated from
rat. The effect was due to the nonspecific interference in calcium in availability needed for the
smooth muscle contraction that resulted in relaxation (Orie,1997).

ANTIOXIDANT ACTIVITY
Vijayakumar et al (2008) reported in rats the flavonoids extracted from Musa paradisiaca
exhibited potent antioxidant activity by stimulating the activities of superoxide dismutase
(SOD)and Catalan which reduced the levels of per oxidation products such as malondialdehyd ,
hydrogen peroxide and conjugated dishes. The antioxidant activities of the aqueous extract of
unripe plantain (Musa paradisiaca)were evaluated and their inhibitory actions on sodium
nitroprusside induced lipid peroxidation in rat pancreas in vitro and also characterized the main
phenolic constituents of the plantain products using gas chromatography analysis (Sidiqat et
al.,2013).
2.5.3 TAXONOMY OF MUSA PARADISIACA
The classification of Musa paradisiaca as reported by Smith(1977).
Kingdom-plantae
Division-Spermatophyta
Sub-division-Angiospermae
Phylum-Tracheophyta
Class-Liliopsida
Order-Zingiberales
Family-Musceae
Genus-Musa
Species- Paradisiaca
Variant- False horn
CHAPTER THREE
3.0
MATERIALS AND METHODS
3.1
CHEMICALS AND REAGENTS
The following chemicals and reagents were used; they were obtained locally in Abeokuta:
xylazine/ketamine and lithium heparin. Hydrochloric acid, tetraoxosulphate (vi) acid, sucrose,
mannose, sorbitol, ethylenediaminetetraacetic acid (EDTA), sodium-adenosine triphosphate (Na2ATP), magnesium chloride, sodium chloride, potassium chloride, and calcium chloride are among
the chemicals that make up the homogenization medium-A. These came from the Carl Roth
GMBH in Karlsruhe, Germany. The purest quality of chemicals and reagents was used for
everything else.
3.2
APPARATUS AND EQUIPMENT
The following are the apparatus and equipment used; Blender, animal cages (plastic, perforated
and wired), dissecting set, dissecting board, beakers, Eppendorf tubes (2ml and 5ml), 10ml
heparinized tubes, centrifuge, homogenizer, UV-visible spectrophotometer, pH meter, analytical
balance, pocket scale, measuring cylinder, cooler, refrigerator, water bath, hot plate, tissue paper,
paper tape, thermometer, micro pipette, cotton wool, rubber bands, needle and syringe, latex
gloves, nose mask, cuvette and micro pipette tips.
3.3 PREPARATION OF BANANA STALK EXTRACT
The plantain stem was purchased from DUFARMS in Abeokuta's FUNAAB. The stalk was
cleaned (of debris) with clean water, peeled, then rinsed once more before being weighed. After
being peeled, the stem was blended with distilled water. In order to eliminate the shaft, the resulting
homogenate was sieved twice with a white handkerchief and three times with a sieve. After leaving
the solution to settle for the night, the sediment was collected. The extract was then produced by
heating the resulting solution in a water boiler at 60°C. The finished stock solution was created by
weighing the extract and reconstituting it with distilled water. The administered concentrations
were then made from the stock solution.
Fig. 3: A picture of the banana stalk used for the research
3.4
PREPARATION OF GUAVA LEAVE EXTRACT (GLE)
On the Abeokuta-Ibadan expressway, a building in the Odo-eran neighborhood of Abeokuta was
used to gather fresh guava leaves. The guava leaves were carefully washed before being allowed
to dry at room temperature until their weight was consistent. Following that, the desiccated leaves
were ground into a powder and sieved. The obtained fine powder was mixed with distilled water
and put through a muslin fabric sieve. The filtrate was refrigerated and given time to settle before
undergoing a second round of muslin fabric filtration and being heated to 600°C to produce the
extract. To create the finished stalk solution, the extract was reconstituted with distilled water.
3.5
EXPERIMENTAL ANIMALS
For the research, 48 male albino rats weighing between 150 and 270g were used. They were
purchased from a respected animal house in Alabata, Abeokuta. Prior to starting the induction of
diabetes, the rodents were given two weeks to acclimate. The animals were housed in ambient
circumstances in well-ventilated cages. They were kept on a typical pelletized diet and unlimited
water.
3.4.1 EXPERIMENTAL INDUCTION OF DIABETES
A single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg body weight) dissolved in a
newly made citrate buffer (0.1 M, pH 4.5) solution made the rats diabetic after an overnight fast.
They received glucose for six hours after receiving STZ to speed up the onset of diabetes. To
validate the onset of diabetes, fasting blood glucose (FBG) levels were measured 72 hours after
STZ injection. Diabetic rats were identified as those with FBG levels above 150mg/dL and were
chosen for the trial.
3.4.2 EXPERIMENTAL DESIGN
Eight groups of forty-eight male albino rats (n=6) were formed before the trial began. To acquire
the baseline data, Group I (the baseline) was sacrificed. The vehicle (citrate buffer) was
administered to Group II, which acted as the control, before they were sacrificed on the third day.
After receiving a single intraperitoneal injection of streptozotocin (50 mg/kg bwt. ), Groups III
and IV were made diabetic and were only provided a basic diet and distilled water for the duration
of the trial before being killed on the third and 28th days, respectively. For 28 days, oral doses of
BSE (100 mg/kg bwt) and GLE (100 mg/kg bwt) were given to Groups V and VI, respectively, to
manage their diabetes. Groups VII and VIII received oral BSE (100 mg/kg) and (300mg/kg)
respectively but were not made diabetic.
The experimental design is represented in Table 1.
Table 1: The experimental design for the study
3.5.
SPLEEN HARVEST DURING SACRIFICE
At the end of the experimental period of each group, the animals were sacrificed after an overnight
fasting except in the case of the baseline .Ketamin/xylazine (0.5ml/250g bwt.) was injected into
the rat as an anesthesia and the animals were pinned to a dissecting board exposing the dorsal part
which was carefully sliced opened to expose the organs. The blood was collected from each the
rat via the abdominal arteries puncture into a heparinized tubed and was centrifuged at 3500 rpm
for 5 minutes. Spleen was harvested and rinsed with ice cold homogenization medium-A to get rid
of the blood, blotted dry and weighed . Thereafter, the organs were stored in a sample bottle for
homogenization and isolation of mitochondria.
3.6
BIOCHEMICALASSAYS
3.5.1. MITOCHONDRIA ISOLATION
Differential centrifugation was used in the mitochondria isolation which involved homogenization
and centrifugation for the separation of several cell fragments.
The Teflon was used in the homogenization of the harvested caecum with addition of 4ml of
homogenization medium-A per gram of caecum after organ blotting. The resulting homogenate
was then transferred into 5ml Eppendorf tubes and centrifuged at 1000g (3000rpm) for 5 minutes
at 4⁰C. Thereafter, the supernatant was decanted into new clean tubes and kept on ice.
The obtained supernatant was then centrifuged at 13000 rpm for 2 minutes using an ultracentrifuge.
Using a pipette, the supernatant was the removed without disturbing the settled mitochondria.
Futhermore, the mitochondria fraction was washed by additing 1ml of homogenization mediumA to re-suspend and the re-centrifuged at 13000 rpm for 2 minutes to eliminate residual cell debris.
Finally, the resulting mitochondria was then re-suspended in 1ml MAITE medium and the
mitochondria yield was calculated.
Calculation of mitochondria yield
Weight of empty 2ml eppendorf tube = W1
Weight of 2ml eppendorf tube + 1ml of MAITE medium = W2
Weight of 2ml Eppendorf tube + 1ml of MAITE + mitochondria = W3
Weight of mitochondria = W3- W2
3.6
BIOCHEMICAL ASSAYS
3.6.1 DETERMINATION OF THE ACTIVITY CALCIUM-MAGNESIUM ATPASE
Ca2+/Mg2+ ATPase was measured using the Hanahan and Ekhelm technique. (1978). 0.05 mM
CaCl2, 3.6 mM MgCl2, 80 mM NaCl, 33 mM KCl, and 80 mM Tris-HCl solution, pH 7.6, were
added to 0.27 ml of buffer and incubated at 440°C for 2 minutes. The tubes holding the buffer
received 25 l of caecum sample, which was then incubated for an additional 10 minutes at the same
temperature.starting the experiment, 5 l of 2.5 mM Na2-ATP was incrementally added into the
tubes, and they were then incubated once more for 10 minutes. After that, 250 l of 10% TCA were
gradually added to halt the reaction. After a quick shake, the containers underwent a 5-minute
centrifugation at 5000 rpm.
The Stewart technique was used to remove 1 ml of supernatant for inorganic phosphate analysis.
(1979). 250 l of supernatant, 250 l of 1.25% ammonium molybdate, and 250 l of 9% ascorbic acid
were pipetted into Eppendorf containers. After 30 minutes of incubation at room temperature, the
resulting solution produced a colored complex, and the absorbance was measured at 660 nm.
Activity of Ca2+/Mg2+ ATPase was then expressed as mgPi/hr/g where Pi was the inorganic
phosphorous produced in the assay. The phosphate standard curve was used to determine how
much inorganic phosphate (Pi) the ATPase produced.
3.6.2 DETERMINATION OF THE ACTIVITY OF MAGNESIUM ATPASE
Mg2+ ATPase activity was measured using the Tsakiris and Deliconstantinos technique. (1984).
500 l of the buffer, which has the following ingredients: 40 mM Tris-HCl buffer, pH 7.4, 5 mM
MgCl2, 80 mM NaCl, 20 mM KCl, and 1 mM digoxin, was measured into eppendorf containers
and incubated for two minutes at 370°C. After 10 minutes at the same temperature, 5 l of the
caecum sample was added to the tubes holding the buffer.Starting the experiment, 5 l of 2.5 mM
Na2-ATP was incrementally added into the tubes, and they were then incubated once more for 10
minutes. Following that, 100 l of 10% TCA was gradually added to halt the reaction. The tubes
were then violently shaken and centrifuged for 5 minutes at 3000 rpm.
The Stewart technique was used to remove 1 ml of supernatant for inorganic phosphate analysis.
(1979). 500 l of supernatant, 500 l of 1.25% ammonium molybdate, and 500 l of 9% ascorbic acid
were pipetted into fresh eppendorf containers. After 30 minutes of incubation at room temperature,
the resulting solution produced a colored complex, and the absorbance was measured at 660 nm.
The inorganic phosphorous released in the assay, Pi, was used to quantify the activity of the Mg2+
ATPase as mgPi/hr/g. The phosphate standard curve was used to determine how much inorganic
phosphate (Pi) the ATPase produced.
3.6.2. DETERMINATION OF THE ACTIVITY OF SODIUM-POTASSIUM ATPASE
First, Total(Na+/K+/Mg2+) ATPase was measured using the Tsakiris and Deliconstantinos
technique. (1984). Eppendorf tubes were filled with 500 ml of buffer, which contained 40 mM
Tris-HCl buffer, pH 7.4, 5 mM MgCl2, 80 mM NaCl, and 20 mM KCl. The containers were then
incubated at 370°C for 2 minutes. After 10 minutes at the same temperature, 5 l of the caecum
sample was added to the tubes holding the buffer.Starting the experiment, 5 l of 2.5 mM Na2-ATP
was incrementally added into the tubes, and they were then incubated once more for 10 minutes.
Following that, 100 l of 10% TCA was gradually added to halt the reaction. The tubes were then
violently shaken and centrifuged for 5 minutes at 3000 rpm.
The Stewart technique was used to remove 1 ml of supernatant for inorganic phosphate analysis.
(1979). 500 l of supernatant, 500 l of 1.25% ammonium molybdate, and 500 l of 9% ascorbic acid
were pipetted into fresh eppendorf containers. After 30 minutes of incubation at room temperature,
the resultant solution produced a colored complex, and the absorbance was recorded at 660 nm.
The overall (Na+, K+, and Mg2+) ATPase activity was then expressed as mgPi/hr/g, where Pi
represented the inorganic phosphorous produced during the assay. The phosphate standard curve
was used to determine how much inorganic phosphate (Pi) the ATPase produced.
Na+/K+ ATPase activity is equal to overall (Na+/K+/Mg2+) ATPase activity minus Mg2+
ATPase activity.
3.7
STATISTICAL ANALYSIS
Data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey test for
multiple comparisons among the groups of rats using Graph Pad Prism program version 8.0.
Obtained results were expressed as mean ± standard error of the mean. P < 0.05 were considered
statistically significant..The Statistical Package for Social Sciences(SPSS), version25.0 was used
CHAPTER FOUR
4.0 RESULTS
4.1 BLOOD GLUCOSE CONCENTRATION OF THE ANIMALS
Four weeks of BSE(100 and 300 mg/kg bwt.) administration caused no significant difference (p
< 0.05) to the fasting blood sugar level of the groups that were not rendered diabetic as compared
to the control group. Induction of diabetes caused a significant increase (x2.4 and x4) (p < 0.05)
to the fasting blood sugar level of the DB confirmed and DB untreated groups respectively when
compared to the control group. However, the treatment with banana stalk extract and guava leaf
extract(100 mg/kg bwt.) reversed significantly the increased fasting blood sugar level (p < 0.05)
600
b
c
400
b
c
200
a
a a
0
DI
AB
ET C
E ON
DB S C TR
UN O N O L
T
DB TRE RO
L
+ A
DB B TE
10 + SE D
0
G 10
30 BS LE 0
0 E A 10
BS L 0
E ON
AL E
O
NE
Blood glucose concentration
(mg/dl)
caused by the diabetic condition.
FIG. 1: Blood glucose concentration of the animals. Results are expressed as mean ± SEM.
Bars labelled with different letters are significantly different (p ˂ 0.05).
4.2 SPLEENIC CA2+/MG2+ ATPASE ACTIVITY OF THE ANIMALS
Induction of diabetes caused a significant decrease (x2.6) (p < 0.05) to the splenic Ca2+/Mg2+
ATPase activity of the DB confirmed and DB untreated groups when compared to the control
group. However, the treatment with banana stalk extract and guava leaf extract(100 mg/kg bwt.)
reversed significantly the decreased spleenic Ca2+/Mg2+ ATPase activity (p < 0.05)
caused by the diabetic condition. Four weeks of BSE(100 and 300 mg/kg) administration caused
no significant difference (p < 0.05) to the spleenic Ca2+/Mg2+ ATPase activity of the groups that
were not rendered diabetic as compared to the control group
FIG 3: Splenic Mitochondria Ca2+/Mg2+ ATPase Activity In The Animals. Bars Are
Labelled With Different Letters Are Significantly Different (P<0.05)
4.3 SPLEENIC NA+/K+ ATPASE ACTIVITY OF THE ANIMALS
Induction of diabetes caused a significant decrease (x2 and x1.8) (p < 0.05) to the spleenic
Na+/K+ ATPase activity of the DB confirmed and DB untreated groups when compared to the
control group. However, the treatment with banana stalk extract and guava leaf extract(100
mg/kg bwt.) reversed significantly the decreased spleenic Na+/K+ ATPase activity (p < 0.05)
caused by the diabetic condition. Four weeks of BSE(100 and 300 mg/kg bwt.) administration
caused no significant difference (p < 0.05) to the splenic Na+/K+ ATPase activity of the groups
that were not rendered diabetic as compared to the control group.
FIG 4: Splenic Mitochondria Na+/K+ ATPase Activity In The Animals. Bars Are Labelled
With Different Letters Are Significantly Different (P<0.05)
4.4 SPLEENIC MG2+ ATPASE ACTIVITY OF THE ANIMALS
The treatment with banana stalk extract and guava leaf extract(100 mg/kg bwt.) reversed
significantly the decreased spleenic Mg2+ATPase activity (p < 0.05) caused by the diabetic
condition. The reversal was x2.4 significant increase when compared to the DB confirmed and
DB untreated groups.. However. Four weeks of BSE(100 and 300 mg/kg bwt.) administration
caused no significant difference (p < 0.05) to the Mg2+ activity of the spleenic ATPase of the
groups that were not rendered diabetic as compared to the control group.
FIG 5: Splenic Mitochondria Mg2+ ATPase Activity In The Animals. Bars Are Labelled
With Different Letters Are Significantly Different (P<0.05)
4.5 BODY WEIGHT GAIN/LOSS OF THE ANIMALS
There was a gradual weight loss in the body weight of the diabetic animals that weren’t treated
which was significantly different (p<0.05) to the control groups. A significant reversal of the
weight(loss) of the groups treated with extract of BSE and GLE (100 mg/kg bwt.) was evident in
comparison to the control groups. Furthermore, groups that were not rendered diabetic but
administered with BSE (100 and 300 mg/kg bwt.) had a significant weight gain with respect to
the control groups.
FIG 5: Body weight difference between day 1 and day 28 of the animals. Results are
expressed as mean ± SEM (n=6). Bars labelled with different letters are significantly
different (p ˂ 0.05).
CONTROL
DB
CONFIRMED
DB
UNTREATED
DB+BSE100
DB+GLE100
100 BSE
ALONE
300 BSE
ALONE
DAY
1
2.67±1
1±1
-36.33±11
4.67±8
-6.67±7
2.00±1
2.33±1
DAY
2
2.17±4
-22.5±10
-1.00±6
-5.00±9
-4.67±2
7.67±1
9.00±3
DAY
3
-19.17±9
-14.67±12
-4.33±6
-3.67±2
0.67±3
2.00±1
3.33±1
DAY
4
-1.00±5
0.00±3
-2.33±4
-10.00±2
-16.67±1
DAY
5
-2.67±4
-9.33±1
-3.00±6
10.00±2
14.33±2
DAY
6
13.00±9
13.33±7
1.67±7
-1.67±2
-.67±1
DAY
7
7.00±4
-2.00±4
-17.67±11
2.00±1
2.00±1
DAY
8
-3.33±4
8.33±7
18.33±2
2.33±2
3.33±1
DAY
9
-1.00±6
-1.00±2
3.00±1
0.00±1
-1.33±1
DAY
10
2.33±6
-2.00±7
1.67±4
1.33±1
2.33±2
DAY
11
0.00±3
-.67±4
-2.67±3
0.67±1
0.67±2
DAY
12
-1.00±2
4.00±4
-6.67±3
3.00±2
1.00±1
DAY
13
2.00±3
-9.00±6
4.67±2
-1.00±2
-2.00±2
DAY
14
-2.67±3
-7.00±9
-7.67±1
-1.33±4
5.67±3
DAY
15
7.67±3
8.33±11
12.67±1
-11.33±2
-6.33±13
DAY
16
3.33±3
4.33±2
0.10±3
12.00±2
13.67±1
DAY
17
-4.00±1
-21.00±11
-1.00±1
1.67±1
-1.67±5
DAY
18
3.00±1
19.67±9
3.33±2
-3.33±1
4.00±4
DAY
19
-2.00±2
4.67±1
1.67±1
1.33±2
4.67±2
DAY
20
1.00±9
-.33±4
-1.33±3
0.69±2
1.33±1
DAY
21
-4.00±6
0.00±1
-0.67±2
0.69±1
0.69±1
DAY
22
-2.00±4
-1.33±4
2.33±3
0.69±1
-1.67±2
DAY
23
-14.67±3
0.67±4
-5.00±6
2.33±5
3.33±1
DAY
24
-12.00±12
-2.00±5
3.33±2
-2.33±4
1.67±1
DAY
25
-15.33±9
-4.33±6
0.00±3
-1.33±1
-1.00±3
DAY
26
-7.00±4
3.67±8
-2.67±2
6.33±1
5.00±3
DAY
27
-4.00±4
-5.00±7
-3.00±3
-1.33±1
-3.33±1
DAY
28
-8.67±8
-6.00±4
-9.67±5
1.00±2
1.67±2
Table 5: Daily weight gain/loss of the animals.Results are expressed as mean ± SEM (n=6).
4.6 SOMATIC INDEX OF THE SPLEEN OF THE ANIMALS
GROUPS
WT. OF SLEEN
BODY WEIGHT
SPLEEN/BODY WEIGHT
BASELINE
1.03
271
0.004
CONTROL
1.64
206
0.007
DB CONFIRM
0.85
199
0.004
DB+ UNTREATED
0.81
223
0.003
DB+ BSE 100
0.95
186
0.005
DB+ GLE 100
0.99
173
0.006
BSE 100 ALONE
0.92
199
0.005
BSE 300 ALONE
1.17
254
0.005
From the result above, the group that has diabetes but left untreated has a very low splenic somatic
index compared to other groups. As a result of diabetes, there is a shrinkage of the spleen in the
animals. Due to the antidiabetic activities of Musa parasidiaca and Psidum guajava, the spleen of
the groups treated with the extracts were more stable at the end of the experiment.
4.7 WATER INTAKE OF THE ANIMALS
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Day 11
Day 12
Day 13
DB
DB
Contr Confirme untreate DB + GLE DB + BSE BSE Alone BSE Alone
ol
d
d
100
100
100
300
30.8
34.14
49.65
45.57
48.84
31.52
12.9
42.47
55.8
50.8
51.61
51.61
27.93
45.73
36.6
44.43
51.29
50.47
51.29
33.16
41
51.41
50.8
48.84
34.14
46.88
60.89
64.52
54.88
32.83
36.75
78.4
73.34
76.28
34.46
28.42
63.54
76.93
76.61
28.25
31.19
66.32
62.56
68.11
37.73
33.97
70.4
53.08
41.65
32.01
26.42
67.62
28.42
44.59
33.65
29.23
66.48
22.05
35.28
25.97
40.52
61.41
21.39
56.84
27.93
27.62
66.4
31.19
17.64
26.46
37.89
Day 14
Day 15
Day 16
Day 17
Day 18
Day 19
Day 20
Day 21
Day 22
Day 23
Day 24
Day 25
Day 26
Day 27
Day 28
Average Water
Intake(mL)
36.62
44.79
64.35
67.6
66.81
63.21
60.87
55.37
54.39
55.7
58.15
49.16
57.82
51.94
48.67
48.67
39.69
23.84
25.97
26.29
27.76
24.34
24.99
20.41
34.46
39.69
41.81
35.77
39.69
18.62
41.49
20.9
38.55
35.28
25.92
43.12
35.28
33.81
34.46
31.85
30.05
24.33
29.97
24.82
28.42
24.82
16.49
24.33
22.37
20.9
30.42
19.92
19.18
21.72
23.03
46.39
46.55
55.37
45.24
29.07
24.82
21.23
47.53
23.68
31.69
43.45
27.44
22.54
32.18
15.68
29.23
31.19
25.64
25.15
26.62
35.44
25.15
58.82
38.50
40.34
30.59
31.47
The table above shows the average water (in mL) of all the groups of animals across the days of
experiment. The group that was left untreated drank more water as a result of diabetic condition
( Polydipsa ). The groups of animals treated with the extracts of Musa parasidiaca and Psidium
guajava drank less water in comparison with the control group.
4.8 FEED INTAKE OF THE ANIMALS
CHAPTER FIVE
5.0
DISCUSSION AND CONCLUSION
5.1
DISCUSSION
Electrolyte issues are frequently encountered in therapeutic settings.They are most frequently
found in hospital populations, where they affect a wide range of patients (from asymptomatic to
severely sick) and are linked to higher rates of morbidity and mortality. The disturbances of
electrolyte homeostasis are commonly seen in subjects from the community. Proprognosis is
associated with community-acquired electrolytedisorders, even if they are minor and persistent.
(Liamis et al., 2012). Electrolyte problems typically have multiple underlying causes. A number
of pathophysiological variables, including nutritional state, gastrointestinal absorption potential,
concurrent acid-base abnormalities, pharmacological agents, other comorbid illnesses (primarily
renal disease), or acute illness, individually or in combination, are important.
Given that the aforementioned variables are frequently present in diabetics, particularly impaired
renal function, mal-absorption syndromes, acid-base disorders, and multidrug regimens, diabetes
mellitus (DM) is one of the diseases with an increased prevalence of electrolyte abnormalities.
(Elisa et al., 2006).Osmotic imbalance brought on by hyperglycemia frequently causes
electrolyte changes in diabetic patients. Such changes can also be brought on by renal diseases,
diuretics, and calcium channel blockers. (Palmer et al., 2015; Amenabar et al., 2009) When
compared to the control subjects, this research revealed that the majority of diabetic patients had
electrolyte disturbances.
When compared to the control groups in our research, STZ-induced diabetes caused a significant
decrease (p> 0.05) in body weight, which is likely the result of muscle wasting. (Jiju et al.,
2013). Additionally, there was a significant decrease in body weight (p> 0.05) in the groups only
given metformin and banana stalk extract, which was brought on by the rats' failure to consume
less food despite having their appetites suppressed. According to earlier research (Cheng et al.,
2006; Yu et al., 2016) and our most recent studies, a substantial improvement (p>0.05) was seen
in the water intake behavior after a three-week oral administration of banana stalk extract and
metformin, respectively, as these conditions are linked to GI upset and diarrhea.
However, treatment with banana stalk extract substantially reversed the effects of diabetes on
body weight and food intake, as well as their respective increases and decreases.The results of
Al-Yassin et al., who reported an increase in diabetic rats' calcium concentration (hypercalcemia)
in Diwaniya-City, Iraq, are consistent with the 40% increase in calcium ion concentration
(hypercalcemia) among the diabetic rats in this research. (Al-Yassin et al., 2009). This may be
brought on by thiazide treatment, hyperparathyroidism, or increased renal calcium reabsorption.
(Liamis et al., 2014; Palmer, 2011). Additionally, severe insulin deficiency and metabolic
acidosis may contribute to increased bone mineral dissolution and re-absorption, decreased bone
formation due to metabolic acidosis, and the occurrence of hypercalcemia in this case.
Dehydration may also be the primary cause of the condition. (Topaloglu et al., 2005). However,
administration of banana stalk extract greatly reduced the hypercalcemia brought on by diabetes.
In this research, diabetic rats had a 62% lower Mg2+ concentration (hypomagnesemia) than
control subjects. In a study, Palmer stated that between 47.7% and 75% of rats with type 2
diabetes have hypomagnesemia. (Palmer, 2011). The decline in magnesium levels in diabetic rats
may be due to a number of factors, including poor dietary intake, gastrointestinal losses, higher
renal losses brought on by the use of diuretics, and recurrent metabolic acidosis. (Palmer, 2015;
Siddiqui et al., 2014).Low Mg2+ levels can also result from increased gastrointestinal Mg2+
losses brought on by diarrhea as a consequence of diabetic autonomic neuropathy.
Additionally, insulin can cause hypomagnesemia by promoting the net movement of Mg2+ from
external to intracellular space. (Paolisso et al., 20066; Matsumura et al., 2000). It's also possible
that insulin-induced hypoglycemia, which increases epinephrine release, is involved. In poorly
controlled diabetic rats, the risk of hypomagnesemia associated with insulin treatment is elevated
because hyperglycemia causes increased renal Mg2+ loss via osmotic diuresis. (Bauza et al.,
2008). However, administration of banana stalk extract substantially reversed the
hypomagnesemia brought on by diabetes.
The correct contraction and relaxation depend on the presence of calcium and magnesium ions.
Our findings show that the calcium and magnesium ion concentrations of the rat groups that
received metformin did not vary significantly (>0.05). These results indicate that metformin does
not affect the amounts of calcium and magnesium ions. One could speculate that metformin
might have a controlling impact on calcium and magnesium signaling. Numerous research
suggest that metformin has various effects on magnesium ions. (Svare, 2009)
In comparison to the control group, the banana stalk extract significantly reduced the calcium ion
concentration over the period of one and three weeks, respectively. Despite the fact that there
was no appreciable difference (p > 0:05) between the control group and the group that received
the extract over the course of three weeks.
In light of the fact that the extract interfered with calcium ion availability, which is necessary for
the smooth muscle contraction that leads to relaxation, these data support the extract's purported
anti-hypertensive activity. (Orie, 1997). Additionally, our findings demonstrate magnesium ion
efflux from the reported decrease in mitochondrial magnesium ion. Extracellular magnesium ion
has been found to act as a calcium ion agonist, decrease intercellular calcium concentration by
binding to calcium channels (T-type Ca channels, Ca voltage-gated channels, etc.), and possibly
also act as an allosteric inhibitor. (Kolte et al., 2014). As a result, the extract modifies the
concentration of calcium and magnesium ions to produce an anti-hypertensive impact.
Our findings regarding K+ concentration revealed a substantial increase (74%) (p 0.05), which
indicated that hyperkalemia was the primary issue. The percentage of rats affected and the mean
amount of this parameter varied between the three diabetic groups when compared to the control
group. Numerous studies have documented elevated potassium levels among diabetic
populations, supporting our finding. Holkar reported a mean figure of 5.73 0.07 in diabetics with
ketoacidosis in India. (Holkar et al., 2014) Shahid found that diabetic rats with stable glycemic
control had higher potassium levels (7.41 1.8) in a Pakistani study. (Shahid et al., 2005).
Reduced glomerular filtration of K+ (due to acute kidney injury and chronic kidney disease) and
many medications that interfere with K+ excretion are linked to diabetes-associated
hyperkalemia, as are redistribution of potassium from intracellular to extracellular compartment
and changes in the Na+/K+ ATPase that maintained the transmembrane gradients of sodium and
potassium (Liamis et al., 2014; Shahid et al., 2005). These include beta blockers, renin inhibitors,
angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, and potassiumsaving diuretics.
However, the condition of hyporeninemic hypo-aldosteronism, which reduces K+ secretion from
the tubules, is the most frequent cause of chronic hyperkalemia in diabetics. (DeFronzo, 1998).
Patients with this syndrome usually have mild to moderate renal insufficiency and asymptomatic
hyperkalemia. However, administration of banana stalk extract greatly reduced the hyperkalemia
brought on by diabetes.
Hyponatremia rates in diabetic rats were comparable to hyperkalemia rates in our research (63%
vs. 74%). The relationship between sodium and potassium levels is considered to be inverse.
(Saito et al., 1999). Sodium levels are diluted as a result of water migration out of the cell
brought on by hyperglycemia.Insulin, diuretics, and hypoglycemic medications typically lower
salt levels. (Liamis et al., 2014; Palmer et al., 2015). Numerous studies found that the sodium
mean level was lower in the diabetic community. (Sharma et al., 2011). Diabetes commonly
causes hyponatremia, but the disease can also start out with hypernatremia. Due to osmotic
diuresis, uncontrolled DM can also cause hypovolemic-hyponatremia. Additionally, acetoacetate
and -hydroxybutyrate, which are ketone bodies, exacerbate renal sodium depletion in diabetic
ketoacidosis and necessitate urinary electrolyte losses (Liamis et al., 2011; Chiasson et al., 2003).
Tolbutamide can also cause hyponatremia by reducing renal free water clearance. (Moses et al.,
2003). However, administration of banana stalk extract substantially reversed the hyponatremia
brought on by diabetes.
. According to reconstitution studies, the Ca2+/Mg2+ ATPase is adequate to support calcium
transport coupled to ATP hydrolysis on its own. The process of muscular contraction and
relaxation, which is crucial to the muscles, is centered on this pump. Calcium must be pumped
by the Ca2+/Mg2+ ATPase from the cytoplasm into the sarcoplasmic reticulum in order for
muscular relaxation to occur. Muscle relaxation depends heavily on the calcium ion gradients
created by Ca2+/Mg2+ ATPase action. (Yeagle, 2016). Thus, increased activity of this enzyme
indicates increased plasma efflux of calcium, which may result in muscle contraction.
5.2
CONCLUSION
This research used aqueous extracts of Musa paradisiaca stalk and Psidium guajava leaf on
male albino rats to examine the effects of diabetes-induced splenic mitochondriopathy and
the potential reversal of these effects. The study demonstrated that untreated diabetesinduced hyperglycemia in rats resulted in mitochondrial dysfunction, which in turn caused a
substantial decline in splenic Ca2+/Mg2+ ATPase, Na+/K+ ATPase, and Mg2+ ATPase as
well as the animals' body weight. BSE (100 mg/kg bwt) and GLE (100 mg/kg bwt) were
administered to reverse the effects of the diabetic state. It was also clear that BSE (100 and
300 mg/kg bwt) had no discernible impact on the assayed ATPases' activity in the nondiabetic rats, but there was a discernible rise in body weight.
REFERENCES
APPENDIX
1. Preparation of 0.5M Tris-HCl: For 100ml solution, 6.06g of Tris was weighed and
adjusted to 7.4 with HCL. The volume was made up to 100ml and stored at 4°C.
2. Preparation of 1M of MgCl2: For 100ml solution, 2g of magnesium chloride was
weighedand dissolved in 60ml of distilled water. It was then made up to 100ml and stored
at 4°C
3. Preparation of Buffer for Ca2+/Mg2+Activity: For one liter, 9.69g of Tris, 0.0038g of
CaCl2, 0.34g of MgCl2, 4.69g of NaCl and 2:46g of KCI were weighed. All these were
dissolved in a little quantity of distilled water. pH was then adjusted to 7.6 using dilute HCI
and made up to 1000ml.
4. Preparation of 2.5mM Na2-ATP: For 10ml, 0.142g was weighed and dissolved in a
littlequantity of distilled water. It was then made up to the 10ml mark.
5. Preparation of 10% Trichloroacetic acid (TCA): For 100ml, 10.0g of TCA was
dissolved in 100ml of distilled water.
6. Preparation 1.25% Ammonium Molybdate: For 500ml solution, 6.25g of
ammoniummolybdate (NH4)6Mo7O24.4H₂O was weighed and dissolved in 500ml of 6.5%
H₂SO4.
7. Preparation of 6.5% H₂SO4: For 500ml solution, 32.5ml of concentrated sulfuric acid
was measuredand little volume of distilled water was added. It was them made up to mark
with 500ml of distilled water. It was stored at room temperature.
8. Preparation of 9% Ascorbic Acid: For 200ml solution,18g of L-ascorbic acid was
weighed,it was then dissolved in 200ml of distilled water and stored at 4°C.
9. Preparation of 0.66M Trichloroacetic Acid (TCA): For 500ml solution, 53.955g of TCA
was weighed and dissolved in 500ml of distilled water.
10. Preparation of 1% HCl: For 50ml of solution, 500μl of concentrated hydrochloric was
mixed with 50ml of distilled water and stored at room temperature.
11. Magnesium
Working
Reagent:Glycine
25mmol/L,
xylidyl
blue
0.5mmol/L,
chloroacetamide 2.6g/L.
12. Calcium
Working
Reagent:2-amino-2-methyl
propan-1-ol,
O-Cresolphthalein
complexone, 8-hydroxyquinoline and hydrochloric acid.
Calculation of Mg2+concentration in the Plasma Sample:
0.1535
0.4165
x 2 = 0.737mg/dL
Calculation of Ca+ concentration in the Plasma Sample:
0.3125
0.5075
x 10.38 = 6.392mg/dL
Calculation of K+ concentration in the Plasma Sample:
0.153
0.310
x 4 = 1.974mEq/L
Calculation of Na+ concentration in the Plasma Sample:
0.621−0.1675
0.621−0.3405
x 150 = 242.51mEq/L
Preparation of Phosphate Standard Curve
A stock solution of 1.0mM K2HPO4was prepared into a conical flask; the test procedure is
summarized below:
TEST TUBE 1.0mM K2HPO4(µl) DISTILLED H2O(µl) AMM. MOLYBDATE (ml) ASCORBIC ACID (ml)
1
2
3
4
5
6
7
8
9
10
20980
40960
60
80
100
120
200
300
400
1000
940
920
900
880
800
700
600
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
The absorbance was read at 660nm after 30 mins of incubation at room temperature. A plot of
absorbance against phosphate concentration was drawn and the concentration of inorganic
phosphate liberated was determined.
1,00
0,90
Absorbance (nm)
0,80
0,70
0,60
0,50
0,40
0,30
0,20
0,10
0,00
0,00
0,05
0,10
0,15
0,20
0,25
0,30
0,35
Concentation of Inorganic Phosphate (µMPi)
0,40
0,45